Treatment of cells harboring latent HIV-provirus with cytokines or phorbol esters results in the activation of transcription factor NF-kappaB, an increase in transcription from the HIV-LTR and a switch to productive infection. These agents also activate the serine/threonine kinase, Raf-1. Since Raf-1 is a necessary component of the mitogenic signaling cascade, acting at least in part by activating expression of genes driven by AP-1 and Ets binding motifs, we tested the ability of Raf-1 to activate transcription from the HIV-LTR. Using a transient cotransfection assay, we have found that Raf-1 activates expression from the HIV-LTR. Mutants that increased the transforming activity of Raf-1, Raf-pm17, and Raf-BXB displayed increased transactivation activity, and a mutant that did not transform, Raf-301, did not transactivate HIV-LTR-driven expression. Analysis of a series of 5' deletion mutants revealed the importance of sequences between nt-120 and nt-91 for activation by Raf-BXB. Point mutations in this region identified the NF-kappaB motif as the Raf- responsive element in the HIV-LTR. The ability of activated Raf-1 to mimic cytokine activation of HIV-LTR expression suggests that the activation state of Raf-1 may be an important determinant of HIV latency.